PROJECT SUMMARY: In the U.S, 91.8 million adults have prediabetes (PreD). High PreD prevalence represents a prominent public health problem as up to 70% of individuals with PreD convert to type 2 diabetes (T2D). Cardiovascular disease (CVD) risk is high among those with PreD and T2D, and nearly $330 billion/year is spent treating T2D. The American Heart Association and American Diabetes Association have thus called for continued investigation into mechanisms connecting health behaviors to pre-clinical cardiometabolic disease (CMD; e.g., PreD). Physical activity (PA) is a key modifiable determinant of good health. Literature suggests PA recommendation adherence is related to a 25-35% dose-dependent reduction in all-cause mortality, with as little as 75 min/week of regular moderate-intensity PA beneficial. Data from the Diabetes Prevention Program has suggested that, among those with PreD, a behavioral intervention emphasizing PA and diet is at least as successful as the drug metformin in preventing T2D. Yet, many individuals do not fully realize the cardiometabolic benefits of PA, and mechanistic pathways linking PA and cardiometabolic improvements remain incompletely understood. The gut microbiome has been posited as a mechanistic intermediate linking heath behaviors, such as PA, to CMD development. Indeed, the gut microbiome may be important in immunological, metabolic, inflammatory, and neurobehavioral processes, some of which might partially explain how health behaviors influence CMD risk. Yet, limited evidence exists characterizing the effect of PA on the human gut microbiome. To date, most research examining the potential effect of regular chronic PA (i.e., exercise) on the gut microbiome has used animal models. Animal studies often reported exercise-related beneficial alterations to microbial community diversity and short chain fatty acid (SCFA)-producing taxa. SCFAs potentially promote increased energy expenditure and are related to a lean phenotype. While there is support for these findings in a limited number of human studies in apparently healthy adults, all but one study employed a randomized design, most studies were completed in small samples, and the metabolic potential of the gut microbiome was rarely assessed. Therefore, we propose a 100-participant randomized controlled 2-arm parallel trial in individuals 30-64 years old who are overweight or obese and have PreD, to examine how 8 weeks of supervised moderate-intensity treadmill walking exercise for 30-45 min 3 times/week alters the human gut microbiome and gut microbe-derived serum SCFAs. We will further evaluate whether any observed changes in the gut microbiome and/or serum SCFAs impact cardiometabolic profile, body weight, and body composition of study participants. Results will inform the design and implementation of future trials to reduce pre-clinical diseases such as PreD in a mechanistically- informed manner. Pursuant with PA-18-720, this research will also provide the opportunity to ?establish specific body composition or other phenotypic features as indicators of risk for obesity, response to its treatment, or its co-morbidities relevant to NIDDK in well characterized?adult patients.?